Magni:Protocols/Digestion

From OpenWetWare

Jump to: navigation, search

Home        Contact        Lab Members        Publications        Research        Internal        Protocols        Data       


Digestion

To perform a double digest with the Roche enzymes, add to a 0.2-ml PCR tube (25 ul total volume):

    • 1000 ng of DNA (if performing the excision of an insert for a ligation, e.g. E-P, X-P, E-S) or
    • 500-700 ng of DNA (if opening a plasmid E-X or S-P for a ligation) or
    • 50-1000 ng of DNA - 200 ng is ok for most cases - (if performing a screening)
  • Milli-Q water up to 20.5 ul
  • 2.5 ul of buffer H
  • 1 ul of the first enzyme
  • 1 ul of the second enzyme

IF THE DNA HAS A LOW CONCENTRATION AND THE REQUIREMENTS ABOVE CANNOT BE MET, USE UP TO 20.5 ul OF DNA

Mix very well and incubate in the thermocycler at 37°C (Digestion program, reaction volume of 25 ul) for 1-4 hours if using the products for a ligation, or for 10-20 minutes if using the products for a screening.

NOTES

  • If in a hurry, digestion time for ligation purpose can be decreased up to 30 min.
  • Sometimes, a triple digest may be necessary. To perform it, set up the following reaction (35 ul total volume) in a 0.2-ml PCR tube:
    • 500-700 ng of DNA (for ligation purpose) or
    • 50-700 ng of DNA - 200 ng is ok for most cases - (if performing a screening)
  • Milli-Q water up to 28.5 ul
  • 3.5 ul of buffer H
  • 1 ul of the first enzyme
  • 1 ul of the second enzyme
  • 1 ul of the third enzyme

IF THE DNA HAS A LOW CONCENTRATION AND THE REQUIREMENTS ABOVE CANNOT BE MET, USE UP TO 28.5 ul OF DNA

Mix very well and incubate in the thermocycler at 37°C (Digestion program, reaction volume of 35 ul) for 1-4 hours if using the products for a ligation, or for 10-20 minutes if using the products for a screening.
Personal tools